Application of Microgravity Environment to Mesenchymal Stem Cells Culture and Cartilage Regeneration

三维（3D）回转器是一种多向重力产生装置，随着时间的推移，它会产生一个平均10- 3g的环境。人体体细胞在微重力环境下的行为尚不清楚，而且是不可预料的，这显示了我们对知识的无知。我们在这里报告了3D-clinostat （CL组）中的人间充质干细胞（hMSCs）比正常1G环境（C组）中的人间充质干细胞（hMSCs）增殖更广泛。FACS结果显示，培养7 d后，CL组cd44 / cd29或cd90 / cd29双阳性的hMSCs数量比C组增加了6倍。两组hMSC的端粒长度在培养期间没有变化，CL和c组均未检测到端粒酶活性。C组ⅱ型胶原、聚集蛋白表达量升高，CL组表达量降低且未见表达。CL组ERK1/2磷酸化升高，p38MAPK受到抑制，与刺激增殖和抑制分化一致。将hMSCs颗粒移植到小鼠股骨髁间窝软骨缺损中，CL组移植7天后形成透明软骨，而C组移植形成含有少量细胞的非软骨组织。这些结果表明，在3D-clinostat中培养的hMSCs广泛增殖，并具有干细胞在移植后保持向透明软骨分化能力的特点，而在1G条件下培养则无法做到这一点。模拟微重力可能有助于干细胞的体外扩增，并可应用于再生医学和发育生物学。

A three-dimensional (3D) clinostat is a device for multi-direction G force generation, which produces an environment with an average of 10-3 G over time. Behavior of human somatic cells under microgravity environment is only poorly understood and is unexpectable showing ignorance of our knowledge. We report here that human mesenchymal stem cells (hMSCs) in the 3D-clinostat (group CL) proliferated more extensively than those in normal 1G environment (group C). FACS showed that the number of hMSCs double-positive for CD 44/CD 29 or CD 90/CD 29 were 6-times higher in group CL as compared with group C after 7 days culture. Telomere length of hMSCs did not change during culture periods in both groups, CL and C. Telomerase activity was undetectable level in all these hMSC cells. The expression levels of type II collagen, aggrecan increased in the group C but decreased and could not be detected in the group CL. Phosphorylation of ERK1/2 was increased and p38MAPK was repressed in the group CL being consistent with stimulated proliferation and inhibited differentiation. When the pellets of hMSCs were explanted into the cartilage defect made in the intercondylar fossa of the mouse femur, the transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed non-cartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat extensively proliferate with characteristics of stem cells keeping their ability to differentiate into hyaline cartilage after transplantation, which cannot be performed by culturing in 1G condition. Simulated microgravity may be useful for the expansion of stem cells in vitro and could be applicable for regeneration medicine and developmental biology.