Enhanced proliferation and differentiation of human mesenchymal stem cells in the gravity-controlled environment.

BACKGROUND: Human bone marrow mesenchymal stem cells (hMSCs) present a promising cell source with the potential to be used for curing various intractable diseases, and it is expected that the development of regenerative medicine employing cell-based therapy would be significantly accelerated when such methods are established. For that, powerful methods for selective growth and differentiation of hMSCs should be developed. METHODS: We developed an efficient method for hMSC proliferation and differentiation into osteoblasts and adipocytes using gravity-controlled environments. RESULTS: The results indicate that the average doubling time of hMSCs cultured in a regular maintenance medium under microgravity conditions (0.001 G) was 1.5 times shorter than that of cells cultured under natural gravity conditions (1.0 G). Furthermore, 99.2% of cells grown in the microgravity environment showed the expression of hMSC markers, as indicated by flow cytometry analysis. Osteogenic and adipogenic differentiation of hMSCs expanded in the microgravity environment was enhanced under microgravity and hypergravity conditions, respectively, as evidenced by the downregulation of hMSC markers and upregulation of osteoblast and adipocyte markers, respectively. Most cells differentiated into osteoblasts in the microgravity environment after 14 days (~80%) and adipocytes in the hypergravity environment after 12 days (~90%). CONCLUSIONS: Our results indicate that hMSC proliferation and selective differentiation into specific cell lineages could be promoted under microgravity or hypergravity conditions, suggesting that cell culture in the gravity-controlled environment is a useful method to obtain cell preparations for potential clinical applications.

In-situ detection based on the biofilm hydrophilicity for environmental biofilm formation.

A biofilm has a unique structure composed of microorganisms, extracellular polymeric substances (EPSs), etc., and it is layered on a substrate in water. In material science, it is important to detect the biofilm formed on a surface to prevent biofouling. EPSs, the major component of the biofilm, mainly consist of polysaccharides, proteins, nucleic acids, and lipids. Because these biomolecules have a variety of hydrophilicities or hydrophobicities, the substrate covered with the biofilm shows different wettability from the initial state. To detect the biofilm formation, this study employed a liquid-squeezing-based wettability assessment method with a simple wettability index: the liquid-squeezed diameter of a smaller value indicates higher wettability. The method is based on the liquid-squeezing behaviour of a liquid that covers sample surfaces when an air-jet is applied. To form the biofilm, polystyrene surfaces were immersed and incubated in a water-circulated bioreactor that had collected microorganisms in ambient air. After the 14-d incubation, good formation of the biofilm on the surfaces was confirmed by staining with crystal violet. Although the contact angles of captive bubbles on the surfaces with the biofilm were unmeasurable, the liquid-squeezing method could distinguish between hydrophilic and hydrophobic initial surfaces with and without biofilm formation using the diameter of the liquid-squeezed area. The surface wettability is expected to be a promising property for in-situ detection of biofilm formation on a macroscopic scale.

Microgravity influences maintenance of the human muscle stem/progenitor cell pool.

Microgravity induces skeletal muscle atrophy; however, the underlying mechanism is not clarified. In particular, the influence of microgravity on human skeletal muscle stem/progenitor cells (SMPCs) is not well understood. In this study, we used induced pluripotent stem cell-derived human SMPCs to investigate the effect of microgravity on maintenance of the stem/progenitor cell pool. Human SMPCs were induced by free-floating spherical aggregation culture, and derivatized-SMPC spheres were maintained in a microgravity condition (10-3 G) for 2 weeks using a clinostat rotation system. Microgravity culture deformed the SMPC spheres, with no signs of apoptosis. The most obvious change from microgravity culture was a significant decrease in the expression level of Pax7 in the SMPC spheres, with reduced numbers of myotubes in adhesion culture. Pax7 expression also decreased in the presence of the proteasome inhibitor MG132, indicating that the proteasomal degradation of Pax7 protein is not critical for its reduced expression in microgravity culture. Moreover, microgravity culture decreased the expression level of tumor necrosis factor receptor-associated factor 6 (TRAF6) and phosphorylation of its downstream molecule extracellular-related kinase (ERK) in SMPC spheres. Therefore, microgravity negatively regulates Pax7 expression in human SMPCs possibly through inhibition of the TRAF6/ERK pathway to consequently dysregulate SMPC pool maintenance. Overall, these results suggest that skeletal muscle atrophy is caused by microgravity-induced exhaustion of the stem cell pool.